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Labile methyl balances for normal humans on various dietary regimens
Labile Methyl Balances for Normal Humans on Various Dietary Regimens S. Harvey Mudd and Jeffery Normal
young
subjects
were
regimens
which
normal tailed
adult
or in
were
were free
and
stable
in
nitrogen
calculated,
cretions
of creatinine,
sine were
measured.
of
subjects
male
diets
outweighed
labile
methyl
the
and
within
and
total
Taking
additional judged
exsacro-
excretions normal intakes into
of
account
methylated
from
groups
novo
On
moiety
methyl number for
was
intake
at was
methyl
used between
at least
converted
to
the average
least
1.5.
curtailed,
under
the
in
homocys-
cycled
of cycles rose to 3.9 females
significant
average
males
is
methyl
diets
homocysteine
For females,
cycles
of
more
normal
being
males
intake
preformed
the
in
and
before
thionine.
formation
of the
neogenesis
in both
methyl
quantitatively
methionine
3.0
labile
experiments,
times
methyl
a role
When
ingestion
teinyl
of
that
play
de is
than these
Choline urinary
curtailed,
moieties.
and close
essentially
the of
as
or
creatine,
on
cur-
remained
Creatinine
groups.
excretions
compounds,
and
it appears normally
and females.
subjects
equilibrium.
were
must
intakes
The
equilibrium,
intakes
values,
and
balance
of sulfur
female dietary
essentially
choline
weights
the zone of nitrogen zone
either
of cystine.
maintained positive
and
on fixed
semisynthetic
methionine
and virtually
to the
male
maintained
R. Poole
number
When the
1.9
cystalabile
average
for males conditions
and em-
ployed.
published
I
N MAMMALS, methionine has several well-recognized metabolic functions. In addition to its roles in protein biosynthesis, methionine serves as the precursor of the sulfur atom of cysteine, the three-carbon propylamine moiety of spermine and spermidine, and the methyl group of a variety of methylated compounds. Some of the pertinent reactions are outlined in Fig. I. The metabolic mobility of the methyl group of methionine led to its being termed biologically labile.’ Dietary choline (and betaine) methyls also form part of the labile methyl pool.’ It is now known that this is due to the ability of betaine (or choline after oxidation to betaine) to donate a methyl group to homocysteine to form methionine’ (Fig. 1). Additional labile methyl groups In this process, one-carbon fragments, are derived by de novo formation.’ originally at the formate or formaldehyde levels of oxidation, are ultimately reduced to form the methyl group of 5methyltetrahydrofolic acid. A cobalamin-dependent methyltransferase catalyzes the transfer of the methyl of 5methyltetrahydrofolate to homocysteine. Either of these enzymic reconversions of homocysteine back to methionine completes a cycle in which the labile methyl of methionine has been transferred to an acceptor molecule, but the homocysteine moiety has been conserved.6 From the Laboratory of General and Comparative Biochemistry, National Institute of Mental Health, and the Digeslive and Hereditary Diseases Branch, National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda. Md. Received for publication November I I, 1974. Reprint requests should be addressed to S. Harvey Mudd, National Institute of Mental Healrh. Bethesda, Md. 20014. ccjI975 by Grune & Stratton. Inc.
Metabolism, Vol. 24, No. 6 (June),1975
721
722
GLVCINE
MUDD AND POOLE
SERINE
. PUTRESCINE lw Spermidinel 5.1~METHVLENETETRAHVDROFOLATE
K 28
S-METHVLTHIOADENOSINE
SPERMDINE /or Spsrmine)
METHhTHI& RIEOSVL -P 631
CVSTAiHlONlNE 5 011 CVSTEINE
METHVLTHlORlBOSE n-KETDBUTVRATE
Fig. 1. Some of the metabolic relationships important to a consideration of labile methylgroup balance. More detailed discussions of these reactions have been published in many places, including for example, references l-4. The homocysteine conservation cycle consists of reactions l-3 and 7 or 8. The transrulfumtion pathway consistsof reactions 1-5.
Whereas the occurrence of the reactions outlined in Fig. 1 is proven by many lines of evidence, the quantitative relationships between them are less clear. For humans, studies of patients genetically deficient in activity of cystathionine synthase (the enzyme catalyzing reaction 4) have shown that methionine sulfur is ultimately disposed of through the transsulfuration pathway to cysteine and homosulfate,7*8 and studies of persons with defective capacities to remethylate cysteine by ‘N-methyltetrahydrofolate have indicated that de novo methylgroup formation plays a role under at least some conditions.’ However, the partitioning of homocysteine either to cystathionine or to methionine at the branch point which determines the balance between transsulfuration and sulfur conservation6 remains to be determined. If this apportionment could be determined, answers would also be provided to the questions of the rate of de novo methyl formation versus utilization of preformed methyl groups and the average number of times a homocysteinyl moiety passes through the conservation cycle before it is converted to cystathionine. Recently, we conducted experiments aimed at determination of the cyst(e)ine requirements of cystathionine synthase-deficient patients.8 As part of these experiments, normal control subjects were maintained on various methionine intakes which sustained nitrogen equilibrium. In the present paper we report measurements of the urinary excretions during these studies of creatinine and various other methylated compounds derived from S-adenosylmethionine. It will be shown that methyl groups excreted in the form of creatinine probably exceed in large measure the combined excretion of all other endogenously formed methylated compounds. Therefore, the results of these studies permit
723
LABILEMETHYL BALANCES
calculation of approximate balances for the labile methyl group and provide tentative answers to the quantitative questions raised above. MATERIALS
AND METHODS
Methods Urinary creatinine was determined by the Jaffe reaction modified according to Chasson et al. lo This modified method yields urinary creatinine values not significantly different from urine Urinary creatine was measured by a specific creatinines measured by more specific methods.““’ enzymatic method based upon the coupled reactions of creatine kinase, pyruvate kinase. and lactic dehydrogenase.‘* Sarcosine was determined by automated amino acid column chromatography.13 Sensitivity for detection of sarcosine was determined by addition of a small amount of the authentic compound prior to analysis of a second aliquot. To search for urinary dimethylglycine, each milliliter of urine was treated with 1.2 mmoles of NaNO, in a final volume of 2.6 ml 1.6 N acetic acid.14 After incubation for 2 hr at 27” C, which led to the almost complete deamination of primary amino acids, the reaction mixture was lyophilized. The syrupy residue was diluted with water and poured through a column of Dowex-50 H +. After a water wash, dimethylglycine was eluted with 3 N NH40H containing 0.01 M mercaptoethanol. The eluate was lyophilized and the residue dissolved in a small volume of dilute formic acid. Aliquots were then subjected to thin-layer or paper chromatography. Dimethylgyicine was located with iodine vapor.15 Sensitivity for detection of dimethylglycine was judged by addi-
Table 1.
Dietary Intakes of Methionine and Choline and Urinary Excretionsof Creatinine and Cmatine on Various Diets Intake
Nitrogen
Methionine
BChlC.Z*
Patient
Sex
RD
M
Diet
(g/24
AN
M
A
Equilibration Experimental
M
FL
JR
A
Equilibration
M
Experimental
A
Experimental
B
B
F
Experimental
A
spt
F
Experimental
A
KR
F
Equilibration Experimental
DW
F
tVolues
f Not
on the
equilibration
in apparent
in the other
f
+0.30
f
B
Equilibration
balances for
+0.63
Equilibration
Experimental
was
0.36 0.23 0.41 0.24 0.30 0.22 0.23 0.27 0.20 0.49 0.32
A
Experimental
*Nitrogen
f f f + f f f f f f f *
A
Experimental F
-l-o.39 +0.74 +0.28 -0.43 +0.31 +0.01 -0.45 -0.15 + 1.09 + 1.32 - 0.24 -0.65
Equilibration
F
MS
0.51 0.45 0.19 0.13
A
Experimental
BHt
AT
-0.52 f +2.30 f 0.0 f -0.54 f
Equilibration Experimental
negative
instance,
measured.
B
experimental
diets
are
nitrogen
no nitrogen
SE)
diets
not
included
balance
balance
9.6 4.7 8.8 4.6 10.5 4.6 7.7 10.7 4.7 7.8 4.4
4.6 0.5 0.5 0.5
4.5
0.2
Excretion
Creatinine
(mm&/24
+ 2.46 f 0.30 +0.22 i 0.38
Equilibration Experimental
hr f
Urinary Choline
5.7 0.4 3.1 0.3 4.6 0.4
Creatine
hr)
19.5 17.7
0.30 0.29
18.5 14.3
0.32 0.24 0.63 0.34
16.1 14.2 13.8 19.6 17.0 17.0
0.4
0.31 0.25 0.20 0.69
11.0
--t
12.5
1.93
7.9
3.6
11.7
0.23
4.6
0.4
10.5
0.29 0.25
8.2
3.2
11.2
4.4
0.3
8.4
0.16
10.5
4.4
11.7
2.17
0.11
8.2
0.1
10.2
0.27
0.10
10.2
2.4
11.7
2.41
0.1
9.5
0.13
8.1
hove been for
(possibly
was measured.
these
published subjects
due to intake
-?
previously.’ because, of food
not
in one
instance,
prescribed
in the
the
subject
diet)
and,
724
MUDD AND POOLE
tion of graded amounts of this compound to samples before chromatography. Authentic dimethylglycine was recovered in excellent yield through this entire procedure. Dietary intakes were calculated according to published values as follows: creatine and creatinine 16; methionine (equilibration diets) ” ; choline (including phospholipid choline). 18S19
Subjects and Experimental
Plan
The subjects were normal volunteers aged 18-24 yr, in good health as indicated by medical histories, physical examinations, and laboratory tests, including urinary amino acid analyses. The subjects were initially placed on constant equilibration diets. These diets were essentially normal, with daily nitrogen intakes set at 10.0-11.2 g. Daily methionine intakes were 8.8-10.7 mmoles for males, and 7.9-10.5 for females (Table I). Urine and stools were collected. Nitrogen analysis were performed by a micro-Kjeldahl method.20 Nitrogen balances were calculated as the differences between measured dietary nitrogen intakes and the sums of urinary and fecal nitrogens. After the subjects had attained nitrogen balance, the diets were changed to essentially isonitrogenous experimental diets consisting of foods and beverages low in methionine and cystine, and a high caloric supplement with trace protein content. Supplemental L-methionine provided most of the methionine intake. Total daily methionine intakes were in the range of 4.4-4.7 mmoles (experimental diet A, Table I), or 7.7-8.2 mmoles (experimental diet B, Table I). Both these experimental diets contained supplemental L-arginine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, and L-valine in the quantities found in 20 g whole-egg protein. Glycine was added to bring the total supplemental amino acid nitrogen (including methionine) to 10.0 g (and thus, the total food nitrogen to 10.4-I 1.2 g) daily. Throughout the study periods, the subjects received 2 mg folic acid twice weekly. While on the experimental diets, the subjects received vitamins and minerals in the form of multivitamin and mineral capsules. Among the components provided by these capsules were daily doses of pyridoxine . HCI, 0.5 mg, and cyanocobalamin, 2 pg. Each experimental period consisted of 6 days after an initial time for adjustment of caloric intake to maintain stable weights. In some instances a patient completed two such experimental periods on different methionine intakes (Table I). Additional details of these diets have been described.8 RESULTS During the studies described here, the subjects remained in slightly positive nitrogen balance, or within the zone of nitrogen equilibrium (which as been defined as a balance which does not deviate from zero by more than +5% of the ) (Table 1). This was true whether the subjects were redaily nitrogen intake 21*22
20-
01
I
I
6
14
I
IO
I
12
I
I1
14
I
I
16
EXPERIMENTAL DAY
I
I6
I
I
20
I
II
22
1
24
fig. 2. Urinary creatinine excretions of a typical male subject on three dietary regimens. The subject was FL (Table 1). He was started on the equilibration diet at day 1. Values are plotted only for the last 5 days on this diet (days 7-11) during which the subject was demonstrated to have come into nitrogen equilibrium. The subject received experimental diet A during days 12-19 and experimental diet g during days 20-25.
LABILE
METHYL
BALANCES
725
ceiving the essentially normal equilibration diets or the experimental diets low in methionine and choline and virtually free of cystine. The daily urinary creatinine excretions of a typical patient are plotted in Fig. 2. The creatinine excretion for the last 5 days on the equilibration diet was 16.1 + 0.1 mmoles (mean f 1 SE). Upon changing to the experimental diet, the creatinine excretion decreased immediately and thereafter remained quite constant. The excretions during these two successive experimental dietary periods were the same within experimental error (14.2 + 0.2 and 13.8 f 0.2). Values for day 25 have not been plotted since the urine collection for this day was known to be incomplete. The creatinine excretion for day 20 was 11.5 mmoles (not plotted in Fig. 2). When the data were analyzed by the method of Grubbsz3 this value was revealed to be a statistical outlier. In view of the relative constancy of the creatinine excretions for other days, it is thought that this low value was probably due to an incomplete urine collection and this, and a few similar outlying values for other patients, were eliminated in calculating the final mean creatinine excretions. The equilibration diet ingested by FL contained a calculated 5.4 mmoles of creatine plus creatinine. Presumably, after cooking of the food, most of the material was present as creatinine. The experimental diets were virtually free of creatine or creatinine. The decrease in the average urinary creatinine of FL upon changing from the equilibration diet to the experimental diets was 2.1 mmoles, or 39% of the calculated decrease in creatine plus creatinine intake. For all subjects the corresponding decreases were 59% + 14% (SE) for the males and 48% + 8% for females. It seems most reasonable to conclude that these portions of the creatine plus creatinine intakes were absorbed and excreted in the urine. The remainder may have been metabolized in the gut or simply failed to be absorbed. According to this analysis, the best available measures of endogenous creatinine formation are provided by the values for urinary creatinine excretions on the experimental diets. Values for dietary methionine intakes and urinary creatinine excretions for all periods during which the subjects were in positive nitrogen balance or within the zone of nitrogen equilibrium are assembled in Table 1. In order to permit a more complete assessment of methyl-group intake and excretion, calculations were made of the dietary intakes of choline, the chief source (in addition to methionine) of ingested labile methyl groups. The values are listed in Table 1. In addition, the excretions of two other methylated compounds, creatine and sarcosine (N-methylglycine), were experimentally determined. These compounds were chosen because the experimental diets contained substantial amounts of glycine. Although increased dietary glycine is known to sometimes increase urinary creatine excretion,24 under our experimental conditions creatine excretions remained rather low (Table 1). Significant amounts of sarcosine were not detected in the urine of any subject, even during the experimental diets. Since the sensitivity of the method used was sufficient to detect approximately 0.2 mmoles sarcosine in a daily urine volume, it was concluded that methyl groups excreted as sarcosine were insignificant in comparison with the methyl excreted as creatinine.
726
MUDD AND POOLE
Table 2. Methionine and Tolal Choline Intakes and Urinary Creatinine and Creatine Excmtions Males Equilibrium
Females
Experimental
A
Experimental
Diet
6
(mm&s/24
Equilibrium
Experimental
A
Experimental
Dietary
methionine
9.9 f 0.4
4.7 f 0.0
7.8 zsz0.1
9.2 i
0.7
4.5 f 0.1
Dietary
choline
4.5 * 0.5
0.4 f 0.0
0.5 + 0.1
3.4
f
0.4
0.4
f
0.1
0.1
f
0.0
Urinary
creatinine
11.6
f
0.1
10.6
+
0.8
9.9
f
0.4
Urinary
creatine
1.3 f
0.6
0.8
f
0.6
0.3
18.4
+
0.8
15.8
f
0.9
15.4
f
1.6
0.4
f
0.1
0.3
+
0.0
0.5
f
0.2
6
hr f- SE)
8.2 zt 0.1
To gain further information about the metabolism of choline, the urine of two male and two female subjects was examined for dimethylglycine. None was detected in samples collected during either equilibration or experimental dietary periods (sensitivity, l-2 mmoles/day). These data are summarized in Table 2, in which are compiled the mean methionine and choline intakes and the mean urinary creatinine and creatine excretions. These data, together with some additional values taken from the literature, provided bases for calculations of labile methyl balances under each of the various dietary regimens. These calculations are set forth in the Discussion. DISCUSSION
During the present experiments both male and female normal young-adult volunteers were maintained on fixed dietary regimens which were either substantially normal or were semisynthetic and curtailed in methionine, choline, and cystine intake. The subjects maintained steady weights,* were in slightly positive nitrogen balance or within the zone of nitrogen equilibrium (Tablel), positive nitrogen balance or within the zone of nitrogen equilibrium (Table 1), and, within experimental error, were close to, or only slightly below, the zone of sulfur equilibrium .8 All these criteria indicate that, as a first approximation, each subject was in a steady-state condition on each diet. Thus, he was presumably experiencing neither a marked net gain nor loss of methyl groups, and the experimental data on methyl-group intake and methyl-group excretion can be used to calculate total body methyl balances. Before attempting to make such calculations, it is necessary to evaluate the extent to which methyl groups were utilized for three purposes not actually measured during our studies. These are (1) formation of additional methylated excretory products; (2) oxidation to one-carbon fragments at the formaldehyde, formate, or CO2 levels of oxidation; and (3) biosynthesis of the polyamines, spermine and spermidine. Additional Methylated Excretory Products Values taken from the literature for most of the well-known and/or quantitatively important methylated products found in the urine of normal humans are listed in Table 3. Creatinine, creatine, and sarcosine are not listed since these were specifically measured during the present experiments. In compiling these values, it was possible in most instances to allow for dietary intakes of these compounds, so that the amounts listed are those synthesized endogenously each
LABILE
METHYL
727
BALANCES
Table 3.
Urinary Excretion of Methylated Compounds by Normal Adult Humans’ Urinary excretion (meq methyl moiety/Z4
Compound
Not
Anserine
References hr)
detected
25 26
0.32T
Cornitine Catechol
amines
Choline
(and
and
18,29,30
0.06
betoine)
Dimethylamineg
0.60
Methionine
0.05
Methyloted
27.28
0.04
derivatives$
oliphotic
basic
amino
31 32.33 34
0.37
acidsg Not
Methylamine
detected
(<
0.05)
35
0.12
36
0.01
37
3-Methylhistidine
0.10
38
1 -Methylhistidine
0.03
38
Methyloted
purine
N-Methylglycine
and
pyrimidine
derivatives11
(sarcosine)
N-Methylnicotinic Trimethylomine
and
trimethylamine
‘Except
30,40
2.33
oxidett
4.38
Tota I
cluded
39
0.18
acid**
in the cases
from
the values
of
methionine
listed.
and
Where
choline,
o ronge
was
the dietary given
contribution
in the originol
of each
publication,
compound the
mean
has been value
ex-
is listed
here. tValue
listed
$ Includes
is for males.
For females,
metonephrine,
0.05.
normetonephrine,
4-hydroxy-3-methoxymandelic
acid,
and
4-hydroxy-3-
methoxyphenylglycol. $Derived
partially
of methylamine.3’ (Includes
by the
action
of intestinal
bacteria
upon
choline
and
lecithin,
partially
by
methylation
(See text.)
NE,NE,NE-trimethyllysine,
NE,NE-dimethyllysine,
NE-methyllysine,
NG,NG-dimethylarginine,
NG,N’G-dimethylorginine. II Includes guonine.
1 -methylodenine,
‘N-methylguanine,
methylguonosine,
6N-methylodenosine, 7-methylguonine,
‘N-methylguanosine,
2’-0-methylcytidine,
‘N-dimethylguanine,
8-hydroxy-7-methylguanine,
’ N-methyl-
‘N-dimethylguonosine,
5-acetamino-6-omino-3-methyluracil,
’ N-
1 -methylhypoxanthine,
l-
methylinosine. **Includes ttMost
N-methylnicotinamide of this material
lecithin.30v40v4’ proboble
dietary
Additional
probably
also. arises
trimethylamine
by the
formed
action upon
of
alkaline
intestinal hydrolysis
bacteria of
upon
urine
dietory
is excluded
choline as
being
and of
origin4
day. Note that these values are in milliequivalents, rather than millimoles, so that these excretions may be used directly to assess the labile methyls consumed in the formation of the compounds listed. Trimethylamine and trimethylamine oxide are exceptional among the compounds listed in Table 3 in that these methylated amkes are thought to be derived not by endogenous synthesis, but rather by the ac’fion of intestinal bacteria upon orally ingested choline and lecithin.30,@,4’ Dimethylamine is formed partially by intestinal bacterial demethylation of trimethylamine and partially by methylation of methylamine which, in turn, is formed from glycine.3’ In the rat, it has been estimated that 25% of the excreted dimethylamine arises from choline.3’ In the absence of further data, this estimate will be used here for humans. In terms of the labile methyl balance, these exceptional cases imply that approximately 0.9 mmoles of choline from a normal diet are de-
720
MUDD
AND POOLE
graded in the gut.* Since there is virtually no choline in normal feces,42 the remainder of the choline is presumably absorbed. On the equilibration diets, our male subjects were therefore absorbing 4.5 - 0.9 = 3.6 mmoles of choline. Since each mole of choline contains three equivalents of methyl moieties, one of which is biologically labile, and two of which are not, our male subjects were absorbing during the equilibration diets 3.6 meq of labile methyl in the form of choline and 7.2 meq of nonlabile methyl. Methyl Group Oxidation
As elucidated by Mackenzie and his colleagues, oxidation may be an important alternative metabolic fate for methyl groups. In the 24 hr after a dose of “CH3-methionine, rats exhaled as 14C02 5%-7% of the administered radioactivity when on a 0.6% methionine diet. Respiratory r4C02 production was sharply increased by additional dietary methionine or choline.43*44Further addition of cystine eliminated the choline-dependent increase. Choline and betaine methyl groups are also oxidized (13% and 32%, respectively, expired as 14C02 within 24 hr.4S Normal human subjects on unspecified diets given intravenous tracer doses of i4CH3-methionine expired 2.0% f 0.3% (SE) of the administered radioactivity as i4C02 in 2 hr.46t Extrapolation suggested that these subjects would have exhaled at least 6.0% + 1.0% of the methyl group of the administered methionine as 14C02 by the end of 24 hr.? Assuming these subjects were taking diets reasonably analogous to the present equilibration diets, our male subjects probably exhaled at least 0.06 x 9.9 = 0.59 mmoles of CO* derived from the methyl group of methionine each 24 hr. The corresponding figure for our female subjects would be 0.55 mmoles. Mackenzie and co-workers also clarified some of the intermediate steps in the oxidation of labile methyl groups. 47The metabolic cycle suggested by Mackenzie and Frisell.48 is reproduced on the left side of Fig. 3. Appropriate parts of this pathway may account for oxidation of the methyl groups of dietary choline or betaine. For methionine methyl, more recent work has suggested a simpler oxidation pathway consisting of the S-adenosylmethionine-dependent methylation of glycine to sarcosine, and the subsequent oxidation of sarcosine by sarcosine dehydrogenase49m5’ (Fig. 3, reactions 2 and 18). Sarcosine dehydrogenase (E.C. 1.5.3.1) converts the methyl group to the oxidation level of formaldehyde.52 Acceptance of this pathway as the major one for the oxidation of methionine methyl groups implies that the appearance of r4C02 after 14CH3methionine administration will furnish only a minimum estimate of the extent of methionine methyl oxidation, since measurements of respiratory i4C02 will not take into account the retention of a major portion of the oxidized methyl group as the P-carbon of serine,52 or in the form of other metabolites, or in
*The amount of choline degraded by intestinal bacteria was calculated as follows: Millimoles of choline degraded to yield a total of 2.33 methyl meq in trimethylamine and trimethylamine oxide (Table 3) = 2.33/3 = 0.78. Millimoles of choline degraded to yield 25% of the dimethylamine excretion of 0.68 methyl meq (Table 3) = (0.68 x 0.25)/2 = 0.09. Total degradation of choline = 0.78 + 0.09 = 0.87 = 0.9 mmoles. tT. Sargent, personal communication to the authors (1974).
LABILE METHYL
729
BALANCES
Reactions Fig. 3. possibly involved in methyl-group oxidations. The lefthand cycle is that proposed by Mackenzie and Frisell.” The particcompounds ipating in the portion of this cycle between serine and choline may be the phosphatidyl derivatives. The righthand cycle is that proposed by glumenstein and Williams?Q
ETHANOLAMINE
METHYL.
0
bodily CO* pools. The extent of oxidation of choline methyl will also be underestimated by measurement of respiratory 14C02 production for much the same reasons, since the one-carbon fragments formed by oxidation of this compound enter the same metabolic pool as those produced by oxidation of methionine methyls (Fig. 3). An alternative index of the rate of methyl-group oxidation is provided by measurements of sarcosine excretion in hypersarcosinemic patients. Such patients are presumed to lack activity of sarcosine dehydrogenase,53 a presumption which has been confirmed by hepatic enzyme assay in one case.54 Both the enzyme data54 and the fact that for this patient, in contrast to the normal,s3 60”/0-80% of sarcosine loads were recovered in the urine in the first 24 hr* indicate that the metabolic block in this patient was complete enough to ensure that she metabolized little sarcosine. Since there is little or no sarcosine in the plants5 or animals6 materials found in normal diets, and since the normal human excretes little, if any, sarcosine (O-0.02 mmole/24 hr),53 the sarcosine excreted by this sarcosine dehydrogenase-deficient patient provides a measure of the amount of sarcosine endogenously formed and subsequently metabolized by sarcosine dehydrogenase in the normal human. At age 6, under basal conditions, the girl in question excreted 5.3-7.9 mmole sarcosine in her daily urine.53 At age 12, a single daily urine contained 2.9 mmole sarcosine.1 The few additional published values for sarcosine excretion in hypersarcosinemic patients are all derived from boys 2 yr old or younger. These values range from 0.9 mmoles/24 hr (age 3 mo) to 5.6 mmoles/24 hr (age 2 yr).37,57,58Although clearly of interest for our present purposes, no data on the effects of variations in methionine, choline, cystine, or glycine intakes on sarcosine excretion have been reported for hypersarcosinemic patients. Thus, the rather fragmentary data presently available suggest that the normal human will probably metabolize in the range of 5 + 3 mmoles of methyl groups daily by this pathway. *T.
Gerritsen,
personal
communication
to the authors
(1974).
730
MUDD
AND POOLE
These oxidized methyl groups may derive from either the labile methyl pool (methionine and one of the choline-betaine methyls) or from the biologically nonlabile methyls of choline-betaine which become the methyl of sarcosine. On the equilibration diets, our male subjects were absorbing approximately 3.6 mmoles of choline. The fate of this compound is not known with certainty, but complete conversion to sarcosine would account for the major portion of the 5 + 3 mmoles of this compound which are expected to be formed and metabolized in the normal human each day. If so, under these dietary conditions, relatively little of the oxidized methyls would have derived from labile methyls, a conclusion which agrees with the results of the measurements of respiratory 14COz which indicate that somewhat more than 0.6 mmoles of methionine methyl were oxidized each day. On the experimental diets, on the other hand, the subjects received many fewer nonlabile dietary methyls (Table 2), so that, if methyl-group oxidation had continued unabated, a large portion would have derived from labile methyls. However, the dietary conditions of decreased methionine and lack of choline are those which, in the rat, decreased the rate of 14C02 production from “CH,-methionine. Thus, it seems probable that total methyl-group oxidation may have been severely curtailed during the experimental diets and that, again, only small amounts of labile methyls would have been accounted for by oxidation.
Pol_vamine Biosynthesis Additional methionine will be utilized for spermine and spermidine biosynthesis. The three-carbon moieties of these polyamines are formed from decarboxylated S-adenosylmethionine. 59 The methyl-containing product of these reactions if 5’-methylthioadenosine, which may subsequently be subjected to phosphorolysis and hydrolysis to yield methylthioribose@’ (Fig. 1). The metabolic fate of this compound is unknown, but its methyl group may be presumed to be the labile methyl pool. In view of these uncertainties as to the methylated excretory product(s) formed as a result of polyamine synthesis, the demand on the methionine pool for polyamine synthesis was estimated from the turnover rate: The total spermine in the adult human body (calculated by use of the concentrations reported and using normal organ weights6*) is somewhat less than by Tabor and Tabor, 4 mmoles. We are not aware of comparable data for spermidine. However, the comparative spermidine and spermine contents of a variety of mammalian tissue@ indicate that the total body spermidine may be of the same order of magnitude as that of spermine. Spermidine of rat liver and brain has been estimated to turn over with a half-life of about 4 days63 or longer.@ If total human body spermidine turns over at a similar rate, approximately 0.5 mmole of methionine would be utilized daily in the course of the biosynthesis of this polyamine. This may be a considerable overestimate, since the human turnover rate is likely to be lower, and since dietary intake of spermidine has not been taken into account. Since the turnover of spermine is markedly slower than that of spermidine,63 the diversion of methionine for spermine biosynthesis is likely to be so low as to be negligible for our present purposes. Thus, the total daily
LABILE
METHYL
731
BALANCES
polyamine synthesis in the adult human may be taken as 0.5 mmole/day or less. There will be a corresponding demand for methionine. In distinction to utilization of methionine for methylation, or for methyl-group oxidation, polyamine synthesis also involves a net loss of the homocysteinyl moiety of methionine. Methionine following this pathway will contribute neither to transmethylation nor to the sulfur conservation cycle. Balances and Cycles
Sufficient data are now available to permit tentative calculations of labile methyl-group balances during the present experiments. The pertinent values are summarized in Table 4. Apparently, for normal humans, the demand for labile methyl groups is determined to a major extent by the requirement for creatine-creatinine formation. Creatinine is formed nonenzymically in proportion to the amount of phosphocreatine and creatine stored in muscle.65 The result is to place upon the normal adult human a relatively large and inflexible demand for labile methyl groups. In males, with larger muscle masses, this demand by itself may outweigh the labile methyls absorbed from an ordinary diet. For females, creatinine formation is less and may possibly be balanced by the Table 4.
Tentative
labile Methyl Ralances on Several Dietary Regimens M&r
Equilibration
Females
Experimental
A
Experimental
Diet
(meq
Equilibr.tion
B lobile
methyl/24
Experimental
A
Experimental
B
hr)
Inputs Methionlne’
9.9
4.7
7.8
9.2
45
Cholinet
3.6
o-o.4
o-o.5
25
o-o.4
Total
13.5
7.8-8.3
4.7-5.1
11 7
a2
I
o-o.
4 5-4.9
8.2-8
3
0”tnowr Creatin8net Other
Polyomine
15.8
15.4
10.3
10.6
1.9
1.8
2.0
2.6
2.1
16
0.5
05 ?
05
0.3
05
>0.6
0.5 ?
>0.6
?
?
18.6
18.1
17.9
14.0
13.2
120
5.1
13.0
9.6
23
8.3
37
1.9
3.9
2.3
I5
3.0
14
ryntherir
Oxidationq Total
15.6
methylations$
occwnted
Mi”irn”rn
for
ertimoter
of average
Methylneagenerirll Cyclea
99
per homocysteinyl
moiety** Per cent homocyr+eine remsthyhtedt
*From tVolues
57
74
33
67
29
2.
from
certainty these
47
Table
Table
2, excluding
in the values
during
the
portion
the experimental
of choline diets
degraded
reflects
by intestinal
uncertainties
bacteria
as to bacterial
(see text).
The
degradation
un-
under
conditions.
$Values present
during
during
perimental
diets
jjlncludes choline,
creatine
(Table
discussion
**Total
2) and
all
or from
Table
for
2. In order
equilibration
to exclude
diets
are
dietory
means
sources
of all
values
of
creatinine
during
the
ex-
methyloted oxide,
compounds
and
the
listed
methyls
of
in
Table
dimethylomine
3,
except arising
for from
methionine, bacterial
in text.
methyl
output
minus
tronsmethylations
-
from
listed
glycine.
+
maximum time-averaged
planation. t t 100
taken
values
trimethylamine
of choline
(IMinimum
diets
diets,
(see text).
trimethylomine,
degrodotion qSee
experimental
equilibration
(1 OO/averoge
No.
cycles).
labile
methyl
intake.
homocysteinyl
moiety
available.
See
text
for
further
ex-
732
MUDD
AND
POOLE
normal labile methyl intake. In view of the quantitative importance of creatine formation as a fate for labile methyl groups, it would be of interest to reinvestigate factors affecting the activity of guanidinoacetate methyltransferase (E.C. 2.1.1.2). The catalytic properties of this enzyme have received little experimental attention since the pioneering studies of Cantoni and his collaborators@ 20 yr ago. A second conclusion which emerges is that when the labile methyls required for methylations in addition to that of guanidinoacetate are taken into account, endogenous formation of methylated compounds must normally exceed the dietary intake of labile methyl groups. Methyl group neogenesis must then normally be required. Conversely, the homocysteine conservation cycle must normally be operative. For example (Table 4), for the male subjects receiving the equilibration diets, 9.9 mmoles of homocysteine moieties were made available daily, of which up to 0.5 mmoles were removed during the course of each 24 hr for polyamine synthesis, Neglecting minor alternative pathways for degradation of methionine and homocysteine, and averaging over time, this left available approximately 9.7 mmoles of homocysteine which cycled sufficiently, so that at least 18.0 mmoles of methylation occurred from S-adenosylmethionine. More transmethylation may have occurred to the extent that oxidation of methionine methyl by way of sarcosine has been underestimated, and that endogenously formed methylated compounds lost in urine or stools were not taken into account. Each homocysteine moiety must then have cycled on the average at least 1.9 times. (Since each of the uncertainties specified here would lead to underestimation of the extent of cycling, the value of 1.9 is a minimal one.) Expressed in another way, under these conditions at any moment the chances were that at least 47% of the available homocysteine was being reconverted to methionine, while the remainder was being diverted to cystathionine.* As a third conclusion, it is clear that when methionine and choline intakes are curtailed, methyl neogenesis becomes the dominant source of labile methyls. Under the particular conditions of experimental diet A, the average homocysteinyl moiety passed through the sulfur conservation cycle at least 3.9 times in the male subjects, and 3 times in the females (Table 4). The present data are compatible with, although they do not prove, the possibility that methionine intake controls methyl neogenesis so that a reduction in methionine ingestion leads to an enhancement of de novo methyl-group formation. Possible mechanisms to mediate such an effect are suggested by the observations that in rat liver S-adenosylmethionine may inhibit the activity of methylenetetrahydrofolate reductase,68 and that dietary methionine may decrease the activity of ‘Nmethyltetrahydrofolate-homocysteine methyltransferase69 and prevent the accumulation of 5N-methyltetrahydrofolate.70 The values summarized in Table 4 are merely first approximations. Improvements could be made by more complete measurements of methylated excretory products and especially by determination of the extent of oxidation of labile
*This value is within the range of 40”-60% of homocysteine converted each cycle, reported as a preliminary estimate for normal rat liver.67
to cystathionine
during
LABILE
METHYL
733
BALANCES
and nonlabile methyls under a variety of conditions. The specific results obtained apply only to the dietary conditions employed and may have been influenced to an unknown extent, for example, by the very low cystine content of the experimental diets. Nevertheless, the present calculations appear to be sufficient to outline the main avenues of labile methyl-group metabolism in the adult human and to provide a point of departure for assessment of the effects of genetic, nutritional, pharmacologic, or hormonal factors which alter the quantitative relationships between these avenues. ACKNOWLEDGMENT The authors wish to thank Dr. G. L. Cantoni for his interest in this work, MS B. Conerly for performing amino acid analyses, MS E. Bou for her help in planning and analyzing the diets. and MS K. Pettigrew
for analyzing
the data for statistical
outliers.
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ffuids.
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Biol
12. Marymont JH Jr, Smith JN, Klotsch BA, Klotsch S: A simple method for urine creatine. Tech Bull Registry Med Technologists 38: I518, 1968
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13. Spackman DH, Stein WH, Moore S: Automatic recording apparatus for use in the chromatography of amino acids. Anal Chem 30:1190-1206, 1958
5. Mudd SH: Biochemical mechanisms in methyl group transfer, in Fishman WH (ed): Metabolic Conjugation and Metabolic Hydrolysis, vol. 3. New York, Academic Press, 1973,
14. Zappia V, Zydek-Cwick YR, Schlenk F: The specificity of S-adenosylmethionine derivatives in methyl transfer reactions. J Biol Chem
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15. Stein von Kamienski E: Untersuchungen uber die fluchtigen amine der pflanzen. I. Methodik der trennung und des nachweises fluchtiger amine. Planta 50:291-314, 1957 16. Beard Metabolism. co.,
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17. Orr ML, Watt BK: Amino acid content of foods. U.S. Dept of Agriculture Home Economics Research Report. No. 4. Washington, DC, U.S. Government Printing Office. 1959 18. Borgl~n NE: On the excretion in urine. Acta Pharmacol Toxicol 3:1-121, 1947
of choline (suppl I)
19. Hardinge MG, Crooks H: Lesser known vitamins in foods. J Am Diet Assoc 38:240-245, 1961 20. Ma TS, Zuazaga G: Micro-Kjeldahl determination of nitrogen. Jnd Eng Chem 14:280-282. 1942 21. Leverton RM. Gram MR, Chaloupka M, Brodovsky E, Mitchell A: The quantitative
734
amino acid requirements of young Threonine. J Nutr 58:59-81, 1956
MlJDD
women.
I.
22. Swendseid ME, Williams I, Dunn MS: Amino acid requirements of young women based on nitrogen balance data. I. The sulfurcontaining amino acids. J Nutr 58:495-505, 1956 23. Grubbs, FE: Procedures for detecting outlying observations in samples. Technometrics 11:1-21, 1969 24. Beard HH, Espenan JK, Pizzolato P: The effect of the ingestion of glycine, with and without urea, upon creatine-creatinine excretion in the rat and man. Am J Physiol 127:716721, 1939 25. Striver CR, Perry TL: Disorders of b-alanine and carnosine metabolism, in Stanbury JB, Wyngaarden JB, Fredrickson DS (eds): The Metabolic Basis of Inherited Disease (ed 3). New York, McGraw-Hill, 1972, p 476 26. Cederblad G, Lindstedt S: Excretion of L-carnitine in man. Clin Chim Acta 33:117-123, 1971 27. Sjoerdsma A, Engelman K, Waldmann TA, Cooperman LH, Hammond WG: Pheochromocytoma: current concepts of diagnosis and treatment. Ann Intern Med 65:1302-1326, I966 28. Karoum F, Anah CO, Ruthven CRJ, Sandler M: Further observations on the gaschromatographic measurement of urinary phenohc and indolic metabolites. Clin Chim Acta 24:341-348, 1969 29. Casky TZ, Mollerstrom J, Sirek OV: Excretion of choline in the urine of diabetic patients. Arch Intern Med 84730-737, 1949 30. De la Huerga J, Popper H: Urinary excretion of choline metabolites following choline administration in normals and patients with hepatobiliary diseases. J Clin Invest 30: 463-470, 195 1 31. Asatoor AM, Simenhoff ML: The origin of urinary dimethylamine. Biochim Biophys Acta +I1:384-392. 1965 32. Stein WH: A chromatographic investigation of the amino acid constituents of normal urine. J Biol Chem 201:45-58, 1953 33. Morrow G III, Barness LA: Combined in homocystinuria. vitamin responsiveness J Pediatr 81:946-954, 1972 34. Kakimoto Y, Akazawa S: Isolation and identification of NG, NG- and NG, N”dimethylarginine, NE-mono-, di-, and triand glucosylgalactosyland methyllysine, galactosyl-&hydroxylysine from human urine. J Biol Chem 245:5751-5758, 1970
AND POOLE
35. Laffler H: Die fluchtigen amine des menschlichen harnes. Z Physiol Chem 232: 259-262, 1935 36. Chheda GB: Purine and pyrimidine derivatives excreted in human urine, in Sober HA (ed): Handbook of Biochemistry. Cleveland, The Chem. Rubber Co., 1970, p G-106 37. Gerritsen T, Waisman HA: Hypersarcosinemia: an inborn error of metabolism. N Engl J Med 275:66-69, 1966 38. Block WD, Hubbard RW, Steele BF: Excretion of histidine and histidine derivatives by human subjects ingesting protein from different sources. J Nutr 85:419-425, 1965 39. Perlzweig WA, Levy ED, Sarett HP: Nicotinic acid derivatives in human urine and their determination. J Biol Chem 136:729-745. 1940 40. Lintzel W: Untersuchungen Uber trimethylammonium basen. III. Trimethylammoniumbasen im menschlichen harm Biochem Z 27312433261, 1934 41. De la Huerga J, Popper H, Steigmann F: Urinary excretion of choline and trimethylamines after intravenous administration of choline in liver diseases. J Lab Clin Med 38: 904910,195! 42. De la Huerga J, Popper H: Factors influencing choline absorption in the intestinal tract. J Clin Invest 31:598-603, 1952 43. Mackenzie CG, Rachele JR, Cross N, Chandler JP, Du Vigneaud V: A study of the rate of oxidation of the methyl group of dietary methionine. J Biol Chem 183:617-626, 1950 44. Mackenzie CG, Du Vigneaud V: Effect of choline and cystine on the oxidation of the methyl group of methionine. J Biol Chem 195: 487-491, 1952 45. Ferger MF, Du Vigneaud V: Oxidation in vivo of the methyl groups of choline, betaine, and dimethyl-+!I-propiothetin. dimethylthetin, J Biol Chem 185:53357, 1950 46. Israelstam DM, Sargent T. Finley NN, Winchell HS, Fish MB, Motto J, Pollycove M, Johnson A: Abnormal methionine metabolism in schizophrenic and depressive states: a preliminary report. J Psychiatr Res 7:185-190, 1970 Mackenzie CG: The 47. Horner WH, biological formation of sarcosine. J Biol Chem 187: 15-22, 1950 48. Mackenzie CC, Frisell WR: The metabolism of dimethylglycine by liver mitochondria. J Biol Chem 232:417-427, 1958 Williams GR: The 49. Blumenstein J, enzymic N-methylation of glycine. Biochem Biophys Res Commun 3:259-263, 1960
LABILE
METHYL
735
BALANCES
50. Kerr SJ: Competing methyltransferase systems. J BioI Chem 247:4248&4252, 1972 51. Heady JE, Kerr SJ: Purification and characterization of glycine N-methyltransferase. J Biol Chem 248:69-72, 1973 52. Mackenzie CG, Abeles RH: Production of active formaldehyde in the mitochondrial oxidation of sarcosine-CDs. J Biol Chem 222: 145-150. 1956 53. Gerritsen T, Waisman HA: Hypersarcosinemia. in Stanbury JB, Wyngaarden JB, Fredrickson DS (eds): The Metabolic Basis of Inherited Disease (ed 3). New York, McGrawHill, 1972, p 459 54. Gerritsen T: Sarcosine dehydrogenase deficiency, the enzyme defect in hypersarcosinemia. Helv Paediatr Acta 27:33-37, 1972 55. Linko P. Alfthan M, Miettinen JK, Virtanen Al: Free sarcosine in reindeermoss (Cladonia silvatica). Acta Chem Stand 7:1310I31 I, 1953 56. Tallan HH, Moore S, Stein WH: Studies on the free amino acids and related compounds in the tissues of the cat. J Biol Chem 211: 927-939, 1954 57. Hagge W, Brodehl J. Gellisen K: Hypersarcosinemia. Pediatr Res 1:409, 1967 58. Scott CR, Clark SH, Teng CC, Swedberg KR: Clinical and cellular studies of sarcosinemia. J Pediatr 77:805-81 I, 1970
61. Tabor
H.
Tabor
spermine, and related I6:245-300, 1964
CW:
amines.
Spermidine.
Pharmacol
Rev
62. Boyd W: A Textbook of Pathology 8). Philadelphia, Lea & Febiger, 1970, p 46. 63. Russell
DH, Medina
VJ, Snyder
(ed
SH: The
dynamics of synthesis and degradation of polyamines in normal and regenerating rat liver and brain. J Biol Chem 245:6732-6738, 1970 64. Snyder
SH, Shaskan
EG, Harik
SI: Poly-
amine disposition in the central nervous system, in Russell DH (ed): Polyamines in Normal and Neoplastic Growth. New York. Raven Press, 1973, p 199 65. Borsook
H, Dubnoff
JW: The hydrolysis
of phosphocreatine and the origin creatinine. J Biol Chem 168:493-510,
of urinary 1947
66. Cantoni GL, Vignos PJ Jr: Enzymatic mechanism of creatine synthesis. J Biol Chem 209:6477659, 1954 67. Finkelstein
JD:
Methionine
metabolism
in mammals: the biochemical basis for homocystinuria. Metabolism 23:387-398, 1974 68. Kutzbach C, Stokstad ELR: Feedback inhibition of methylene-tetrahydrofolate reductase in rat liver by S-adenosylmethionine. Biochim Biophys Acta 139:217-220, 1967 69. Kutzbach
C, Galloway
E, Stokstad
ELR:
59. Tabor H, Rosenthal SM, Tabor CW: The biosynthesis of spermidine and spermine from putrescine and methionine. J Biol Chem 233: 907-914, 1958
Influence of vitamin B,z and methionine on levels of folic acid compounds and folate enzymes in rat liver. Proc Sot Exp Biol Med 124: 801-805, 1967
60. Pegg AE, Williams-Ashman HG: Phosphate-stimulated breakdown of 5’-methylthioadenosine by rat ventral prostate. Biochem J 115:241-247. 1969
ELR: The effect of methionine on and histidine metabolism in perfused
70. Buehring,
Biochim
Biophys
KU,
Batra
KK,
Acta 2791498~512,
Stokstad folic acid rat liver. 1972
young
subjects
were
regimens
which
normal tailed
adult
or in
were
were free
and
stable
in
nitrogen
calculated,
cretions
of creatinine,
sine were
measured.
of
subjects
male
diets
outweighed
labile
methyl
the
and
within
and
total
Taking
additional judged
exsacro-
excretions normal intakes into
of
account
methylated
from
groups
novo
On
moiety
methyl number for
was
intake
at was
methyl
used between
at least
converted
to
the average
least
1.5.
curtailed,
under
the
in
homocys-
cycled
of cycles rose to 3.9 females
significant
average
males
is
methyl
diets
homocysteine
For females,
cycles
of
more
normal
being
males
intake
preformed
the
in
and
before
thionine.
formation
of the
neogenesis
in both
methyl
quantitatively
methionine
3.0
labile
experiments,
times
methyl
a role
When
ingestion
teinyl
of
that
play
de is
than these
Choline urinary
curtailed,
moieties.
and close
essentially
the of
as
or
creatine,
on
cur-
remained
Creatinine
groups.
excretions
compounds,
and
it appears normally
and females.
subjects
equilibrium.
were
must
intakes
The
equilibrium,
intakes
values,
and
balance
of sulfur
female dietary
essentially
choline
weights
the zone of nitrogen zone
either
of cystine.
maintained positive
and
on fixed
semisynthetic
methionine
and virtually
to the
male
maintained
R. Poole
number
When the
1.9
cystalabile
average
for males conditions
and em-
ployed.
published
I
N MAMMALS, methionine has several well-recognized metabolic functions. In addition to its roles in protein biosynthesis, methionine serves as the precursor of the sulfur atom of cysteine, the three-carbon propylamine moiety of spermine and spermidine, and the methyl group of a variety of methylated compounds. Some of the pertinent reactions are outlined in Fig. I. The metabolic mobility of the methyl group of methionine led to its being termed biologically labile.’ Dietary choline (and betaine) methyls also form part of the labile methyl pool.’ It is now known that this is due to the ability of betaine (or choline after oxidation to betaine) to donate a methyl group to homocysteine to form methionine’ (Fig. 1). Additional labile methyl groups In this process, one-carbon fragments, are derived by de novo formation.’ originally at the formate or formaldehyde levels of oxidation, are ultimately reduced to form the methyl group of 5methyltetrahydrofolic acid. A cobalamin-dependent methyltransferase catalyzes the transfer of the methyl of 5methyltetrahydrofolate to homocysteine. Either of these enzymic reconversions of homocysteine back to methionine completes a cycle in which the labile methyl of methionine has been transferred to an acceptor molecule, but the homocysteine moiety has been conserved.6 From the Laboratory of General and Comparative Biochemistry, National Institute of Mental Health, and the Digeslive and Hereditary Diseases Branch, National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda. Md. Received for publication November I I, 1974. Reprint requests should be addressed to S. Harvey Mudd, National Institute of Mental Healrh. Bethesda, Md. 20014. ccjI975 by Grune & Stratton. Inc.
Metabolism, Vol. 24, No. 6 (June),1975
721
722
GLVCINE
MUDD AND POOLE
SERINE
. PUTRESCINE lw Spermidinel 5.1~METHVLENETETRAHVDROFOLATE
K 28
S-METHVLTHIOADENOSINE
SPERMDINE /or Spsrmine)
METHhTHI& RIEOSVL -P 631
CVSTAiHlONlNE 5 011 CVSTEINE
METHVLTHlORlBOSE n-KETDBUTVRATE
Fig. 1. Some of the metabolic relationships important to a consideration of labile methylgroup balance. More detailed discussions of these reactions have been published in many places, including for example, references l-4. The homocysteine conservation cycle consists of reactions l-3 and 7 or 8. The transrulfumtion pathway consistsof reactions 1-5.
Whereas the occurrence of the reactions outlined in Fig. 1 is proven by many lines of evidence, the quantitative relationships between them are less clear. For humans, studies of patients genetically deficient in activity of cystathionine synthase (the enzyme catalyzing reaction 4) have shown that methionine sulfur is ultimately disposed of through the transsulfuration pathway to cysteine and homosulfate,7*8 and studies of persons with defective capacities to remethylate cysteine by ‘N-methyltetrahydrofolate have indicated that de novo methylgroup formation plays a role under at least some conditions.’ However, the partitioning of homocysteine either to cystathionine or to methionine at the branch point which determines the balance between transsulfuration and sulfur conservation6 remains to be determined. If this apportionment could be determined, answers would also be provided to the questions of the rate of de novo methyl formation versus utilization of preformed methyl groups and the average number of times a homocysteinyl moiety passes through the conservation cycle before it is converted to cystathionine. Recently, we conducted experiments aimed at determination of the cyst(e)ine requirements of cystathionine synthase-deficient patients.8 As part of these experiments, normal control subjects were maintained on various methionine intakes which sustained nitrogen equilibrium. In the present paper we report measurements of the urinary excretions during these studies of creatinine and various other methylated compounds derived from S-adenosylmethionine. It will be shown that methyl groups excreted in the form of creatinine probably exceed in large measure the combined excretion of all other endogenously formed methylated compounds. Therefore, the results of these studies permit
723
LABILEMETHYL BALANCES
calculation of approximate balances for the labile methyl group and provide tentative answers to the quantitative questions raised above. MATERIALS
AND METHODS
Methods Urinary creatinine was determined by the Jaffe reaction modified according to Chasson et al. lo This modified method yields urinary creatinine values not significantly different from urine Urinary creatine was measured by a specific creatinines measured by more specific methods.““’ enzymatic method based upon the coupled reactions of creatine kinase, pyruvate kinase. and lactic dehydrogenase.‘* Sarcosine was determined by automated amino acid column chromatography.13 Sensitivity for detection of sarcosine was determined by addition of a small amount of the authentic compound prior to analysis of a second aliquot. To search for urinary dimethylglycine, each milliliter of urine was treated with 1.2 mmoles of NaNO, in a final volume of 2.6 ml 1.6 N acetic acid.14 After incubation for 2 hr at 27” C, which led to the almost complete deamination of primary amino acids, the reaction mixture was lyophilized. The syrupy residue was diluted with water and poured through a column of Dowex-50 H +. After a water wash, dimethylglycine was eluted with 3 N NH40H containing 0.01 M mercaptoethanol. The eluate was lyophilized and the residue dissolved in a small volume of dilute formic acid. Aliquots were then subjected to thin-layer or paper chromatography. Dimethylgyicine was located with iodine vapor.15 Sensitivity for detection of dimethylglycine was judged by addi-
Table 1.
Dietary Intakes of Methionine and Choline and Urinary Excretionsof Creatinine and Cmatine on Various Diets Intake
Nitrogen
Methionine
BChlC.Z*
Patient
Sex
RD
M
Diet
(g/24
AN
M
A
Equilibration Experimental
M
FL
JR
A
Equilibration
M
Experimental
A
Experimental
B
B
F
Experimental
A
spt
F
Experimental
A
KR
F
Equilibration Experimental
DW
F
tVolues
f Not
on the
equilibration
in apparent
in the other
f
+0.30
f
B
Equilibration
balances for
+0.63
Equilibration
Experimental
was
0.36 0.23 0.41 0.24 0.30 0.22 0.23 0.27 0.20 0.49 0.32
A
Experimental
*Nitrogen
f f f + f f f f f f f *
A
Experimental F
-l-o.39 +0.74 +0.28 -0.43 +0.31 +0.01 -0.45 -0.15 + 1.09 + 1.32 - 0.24 -0.65
Equilibration
F
MS
0.51 0.45 0.19 0.13
A
Experimental
BHt
AT
-0.52 f +2.30 f 0.0 f -0.54 f
Equilibration Experimental
negative
instance,
measured.
B
experimental
diets
are
nitrogen
no nitrogen
SE)
diets
not
included
balance
balance
9.6 4.7 8.8 4.6 10.5 4.6 7.7 10.7 4.7 7.8 4.4
4.6 0.5 0.5 0.5
4.5
0.2
Excretion
Creatinine
(mm&/24
+ 2.46 f 0.30 +0.22 i 0.38
Equilibration Experimental
hr f
Urinary Choline
5.7 0.4 3.1 0.3 4.6 0.4
Creatine
hr)
19.5 17.7
0.30 0.29
18.5 14.3
0.32 0.24 0.63 0.34
16.1 14.2 13.8 19.6 17.0 17.0
0.4
0.31 0.25 0.20 0.69
11.0
--t
12.5
1.93
7.9
3.6
11.7
0.23
4.6
0.4
10.5
0.29 0.25
8.2
3.2
11.2
4.4
0.3
8.4
0.16
10.5
4.4
11.7
2.17
0.11
8.2
0.1
10.2
0.27
0.10
10.2
2.4
11.7
2.41
0.1
9.5
0.13
8.1
hove been for
(possibly
was measured.
these
published subjects
due to intake
-?
previously.’ because, of food
not
in one
instance,
prescribed
in the
the
subject
diet)
and,
724
MUDD AND POOLE
tion of graded amounts of this compound to samples before chromatography. Authentic dimethylglycine was recovered in excellent yield through this entire procedure. Dietary intakes were calculated according to published values as follows: creatine and creatinine 16; methionine (equilibration diets) ” ; choline (including phospholipid choline). 18S19
Subjects and Experimental
Plan
The subjects were normal volunteers aged 18-24 yr, in good health as indicated by medical histories, physical examinations, and laboratory tests, including urinary amino acid analyses. The subjects were initially placed on constant equilibration diets. These diets were essentially normal, with daily nitrogen intakes set at 10.0-11.2 g. Daily methionine intakes were 8.8-10.7 mmoles for males, and 7.9-10.5 for females (Table I). Urine and stools were collected. Nitrogen analysis were performed by a micro-Kjeldahl method.20 Nitrogen balances were calculated as the differences between measured dietary nitrogen intakes and the sums of urinary and fecal nitrogens. After the subjects had attained nitrogen balance, the diets were changed to essentially isonitrogenous experimental diets consisting of foods and beverages low in methionine and cystine, and a high caloric supplement with trace protein content. Supplemental L-methionine provided most of the methionine intake. Total daily methionine intakes were in the range of 4.4-4.7 mmoles (experimental diet A, Table I), or 7.7-8.2 mmoles (experimental diet B, Table I). Both these experimental diets contained supplemental L-arginine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, and L-valine in the quantities found in 20 g whole-egg protein. Glycine was added to bring the total supplemental amino acid nitrogen (including methionine) to 10.0 g (and thus, the total food nitrogen to 10.4-I 1.2 g) daily. Throughout the study periods, the subjects received 2 mg folic acid twice weekly. While on the experimental diets, the subjects received vitamins and minerals in the form of multivitamin and mineral capsules. Among the components provided by these capsules were daily doses of pyridoxine . HCI, 0.5 mg, and cyanocobalamin, 2 pg. Each experimental period consisted of 6 days after an initial time for adjustment of caloric intake to maintain stable weights. In some instances a patient completed two such experimental periods on different methionine intakes (Table I). Additional details of these diets have been described.8 RESULTS During the studies described here, the subjects remained in slightly positive nitrogen balance, or within the zone of nitrogen equilibrium (which as been defined as a balance which does not deviate from zero by more than +5% of the ) (Table 1). This was true whether the subjects were redaily nitrogen intake 21*22
20-
01
I
I
6
14
I
IO
I
12
I
I1
14
I
I
16
EXPERIMENTAL DAY
I
I6
I
I
20
I
II
22
1
24
fig. 2. Urinary creatinine excretions of a typical male subject on three dietary regimens. The subject was FL (Table 1). He was started on the equilibration diet at day 1. Values are plotted only for the last 5 days on this diet (days 7-11) during which the subject was demonstrated to have come into nitrogen equilibrium. The subject received experimental diet A during days 12-19 and experimental diet g during days 20-25.
LABILE
METHYL
BALANCES
725
ceiving the essentially normal equilibration diets or the experimental diets low in methionine and choline and virtually free of cystine. The daily urinary creatinine excretions of a typical patient are plotted in Fig. 2. The creatinine excretion for the last 5 days on the equilibration diet was 16.1 + 0.1 mmoles (mean f 1 SE). Upon changing to the experimental diet, the creatinine excretion decreased immediately and thereafter remained quite constant. The excretions during these two successive experimental dietary periods were the same within experimental error (14.2 + 0.2 and 13.8 f 0.2). Values for day 25 have not been plotted since the urine collection for this day was known to be incomplete. The creatinine excretion for day 20 was 11.5 mmoles (not plotted in Fig. 2). When the data were analyzed by the method of Grubbsz3 this value was revealed to be a statistical outlier. In view of the relative constancy of the creatinine excretions for other days, it is thought that this low value was probably due to an incomplete urine collection and this, and a few similar outlying values for other patients, were eliminated in calculating the final mean creatinine excretions. The equilibration diet ingested by FL contained a calculated 5.4 mmoles of creatine plus creatinine. Presumably, after cooking of the food, most of the material was present as creatinine. The experimental diets were virtually free of creatine or creatinine. The decrease in the average urinary creatinine of FL upon changing from the equilibration diet to the experimental diets was 2.1 mmoles, or 39% of the calculated decrease in creatine plus creatinine intake. For all subjects the corresponding decreases were 59% + 14% (SE) for the males and 48% + 8% for females. It seems most reasonable to conclude that these portions of the creatine plus creatinine intakes were absorbed and excreted in the urine. The remainder may have been metabolized in the gut or simply failed to be absorbed. According to this analysis, the best available measures of endogenous creatinine formation are provided by the values for urinary creatinine excretions on the experimental diets. Values for dietary methionine intakes and urinary creatinine excretions for all periods during which the subjects were in positive nitrogen balance or within the zone of nitrogen equilibrium are assembled in Table 1. In order to permit a more complete assessment of methyl-group intake and excretion, calculations were made of the dietary intakes of choline, the chief source (in addition to methionine) of ingested labile methyl groups. The values are listed in Table 1. In addition, the excretions of two other methylated compounds, creatine and sarcosine (N-methylglycine), were experimentally determined. These compounds were chosen because the experimental diets contained substantial amounts of glycine. Although increased dietary glycine is known to sometimes increase urinary creatine excretion,24 under our experimental conditions creatine excretions remained rather low (Table 1). Significant amounts of sarcosine were not detected in the urine of any subject, even during the experimental diets. Since the sensitivity of the method used was sufficient to detect approximately 0.2 mmoles sarcosine in a daily urine volume, it was concluded that methyl groups excreted as sarcosine were insignificant in comparison with the methyl excreted as creatinine.
726
MUDD AND POOLE
Table 2. Methionine and Tolal Choline Intakes and Urinary Creatinine and Creatine Excmtions Males Equilibrium
Females
Experimental
A
Experimental
Diet
6
(mm&s/24
Equilibrium
Experimental
A
Experimental
Dietary
methionine
9.9 f 0.4
4.7 f 0.0
7.8 zsz0.1
9.2 i
0.7
4.5 f 0.1
Dietary
choline
4.5 * 0.5
0.4 f 0.0
0.5 + 0.1
3.4
f
0.4
0.4
f
0.1
0.1
f
0.0
Urinary
creatinine
11.6
f
0.1
10.6
+
0.8
9.9
f
0.4
Urinary
creatine
1.3 f
0.6
0.8
f
0.6
0.3
18.4
+
0.8
15.8
f
0.9
15.4
f
1.6
0.4
f
0.1
0.3
+
0.0
0.5
f
0.2
6
hr f- SE)
8.2 zt 0.1
To gain further information about the metabolism of choline, the urine of two male and two female subjects was examined for dimethylglycine. None was detected in samples collected during either equilibration or experimental dietary periods (sensitivity, l-2 mmoles/day). These data are summarized in Table 2, in which are compiled the mean methionine and choline intakes and the mean urinary creatinine and creatine excretions. These data, together with some additional values taken from the literature, provided bases for calculations of labile methyl balances under each of the various dietary regimens. These calculations are set forth in the Discussion. DISCUSSION
During the present experiments both male and female normal young-adult volunteers were maintained on fixed dietary regimens which were either substantially normal or were semisynthetic and curtailed in methionine, choline, and cystine intake. The subjects maintained steady weights,* were in slightly positive nitrogen balance or within the zone of nitrogen equilibrium (Tablel), positive nitrogen balance or within the zone of nitrogen equilibrium (Table 1), and, within experimental error, were close to, or only slightly below, the zone of sulfur equilibrium .8 All these criteria indicate that, as a first approximation, each subject was in a steady-state condition on each diet. Thus, he was presumably experiencing neither a marked net gain nor loss of methyl groups, and the experimental data on methyl-group intake and methyl-group excretion can be used to calculate total body methyl balances. Before attempting to make such calculations, it is necessary to evaluate the extent to which methyl groups were utilized for three purposes not actually measured during our studies. These are (1) formation of additional methylated excretory products; (2) oxidation to one-carbon fragments at the formaldehyde, formate, or CO2 levels of oxidation; and (3) biosynthesis of the polyamines, spermine and spermidine. Additional Methylated Excretory Products Values taken from the literature for most of the well-known and/or quantitatively important methylated products found in the urine of normal humans are listed in Table 3. Creatinine, creatine, and sarcosine are not listed since these were specifically measured during the present experiments. In compiling these values, it was possible in most instances to allow for dietary intakes of these compounds, so that the amounts listed are those synthesized endogenously each
LABILE
METHYL
727
BALANCES
Table 3.
Urinary Excretion of Methylated Compounds by Normal Adult Humans’ Urinary excretion (meq methyl moiety/Z4
Compound
Not
Anserine
References hr)
detected
25 26
0.32T
Cornitine Catechol
amines
Choline
(and
and
18,29,30
0.06
betoine)
Dimethylamineg
0.60
Methionine
0.05
Methyloted
27.28
0.04
derivatives$
oliphotic
basic
amino
31 32.33 34
0.37
acidsg Not
Methylamine
detected
(<
0.05)
35
0.12
36
0.01
37
3-Methylhistidine
0.10
38
1 -Methylhistidine
0.03
38
Methyloted
purine
N-Methylglycine
and
pyrimidine
derivatives11
(sarcosine)
N-Methylnicotinic Trimethylomine
and
trimethylamine
‘Except
30,40
2.33
oxidett
4.38
Tota I
cluded
39
0.18
acid**
in the cases
from
the values
of
methionine
listed.
and
Where
choline,
o ronge
was
the dietary given
contribution
in the originol
of each
publication,
compound the
mean
has been value
ex-
is listed
here. tValue
listed
$ Includes
is for males.
For females,
metonephrine,
0.05.
normetonephrine,
4-hydroxy-3-methoxymandelic
acid,
and
4-hydroxy-3-
methoxyphenylglycol. $Derived
partially
of methylamine.3’ (Includes
by the
action
of intestinal
bacteria
upon
choline
and
lecithin,
partially
by
methylation
(See text.)
NE,NE,NE-trimethyllysine,
NE,NE-dimethyllysine,
NE-methyllysine,
NG,NG-dimethylarginine,
NG,N’G-dimethylorginine. II Includes guonine.
1 -methylodenine,
‘N-methylguanine,
methylguonosine,
6N-methylodenosine, 7-methylguonine,
‘N-methylguanosine,
2’-0-methylcytidine,
‘N-dimethylguanine,
8-hydroxy-7-methylguanine,
’ N-methyl-
‘N-dimethylguonosine,
5-acetamino-6-omino-3-methyluracil,
’ N-
1 -methylhypoxanthine,
l-
methylinosine. **Includes ttMost
N-methylnicotinamide of this material
lecithin.30v40v4’ proboble
dietary
Additional
probably
also. arises
trimethylamine
by the
formed
action upon
of
alkaline
intestinal hydrolysis
bacteria of
upon
urine
dietory
is excluded
choline as
being
and of
origin4
day. Note that these values are in milliequivalents, rather than millimoles, so that these excretions may be used directly to assess the labile methyls consumed in the formation of the compounds listed. Trimethylamine and trimethylamine oxide are exceptional among the compounds listed in Table 3 in that these methylated amkes are thought to be derived not by endogenous synthesis, but rather by the ac’fion of intestinal bacteria upon orally ingested choline and lecithin.30,@,4’ Dimethylamine is formed partially by intestinal bacterial demethylation of trimethylamine and partially by methylation of methylamine which, in turn, is formed from glycine.3’ In the rat, it has been estimated that 25% of the excreted dimethylamine arises from choline.3’ In the absence of further data, this estimate will be used here for humans. In terms of the labile methyl balance, these exceptional cases imply that approximately 0.9 mmoles of choline from a normal diet are de-
720
MUDD
AND POOLE
graded in the gut.* Since there is virtually no choline in normal feces,42 the remainder of the choline is presumably absorbed. On the equilibration diets, our male subjects were therefore absorbing 4.5 - 0.9 = 3.6 mmoles of choline. Since each mole of choline contains three equivalents of methyl moieties, one of which is biologically labile, and two of which are not, our male subjects were absorbing during the equilibration diets 3.6 meq of labile methyl in the form of choline and 7.2 meq of nonlabile methyl. Methyl Group Oxidation
As elucidated by Mackenzie and his colleagues, oxidation may be an important alternative metabolic fate for methyl groups. In the 24 hr after a dose of “CH3-methionine, rats exhaled as 14C02 5%-7% of the administered radioactivity when on a 0.6% methionine diet. Respiratory r4C02 production was sharply increased by additional dietary methionine or choline.43*44Further addition of cystine eliminated the choline-dependent increase. Choline and betaine methyl groups are also oxidized (13% and 32%, respectively, expired as 14C02 within 24 hr.4S Normal human subjects on unspecified diets given intravenous tracer doses of i4CH3-methionine expired 2.0% f 0.3% (SE) of the administered radioactivity as i4C02 in 2 hr.46t Extrapolation suggested that these subjects would have exhaled at least 6.0% + 1.0% of the methyl group of the administered methionine as 14C02 by the end of 24 hr.? Assuming these subjects were taking diets reasonably analogous to the present equilibration diets, our male subjects probably exhaled at least 0.06 x 9.9 = 0.59 mmoles of CO* derived from the methyl group of methionine each 24 hr. The corresponding figure for our female subjects would be 0.55 mmoles. Mackenzie and co-workers also clarified some of the intermediate steps in the oxidation of labile methyl groups. 47The metabolic cycle suggested by Mackenzie and Frisell.48 is reproduced on the left side of Fig. 3. Appropriate parts of this pathway may account for oxidation of the methyl groups of dietary choline or betaine. For methionine methyl, more recent work has suggested a simpler oxidation pathway consisting of the S-adenosylmethionine-dependent methylation of glycine to sarcosine, and the subsequent oxidation of sarcosine by sarcosine dehydrogenase49m5’ (Fig. 3, reactions 2 and 18). Sarcosine dehydrogenase (E.C. 1.5.3.1) converts the methyl group to the oxidation level of formaldehyde.52 Acceptance of this pathway as the major one for the oxidation of methionine methyl groups implies that the appearance of r4C02 after 14CH3methionine administration will furnish only a minimum estimate of the extent of methionine methyl oxidation, since measurements of respiratory i4C02 will not take into account the retention of a major portion of the oxidized methyl group as the P-carbon of serine,52 or in the form of other metabolites, or in
*The amount of choline degraded by intestinal bacteria was calculated as follows: Millimoles of choline degraded to yield a total of 2.33 methyl meq in trimethylamine and trimethylamine oxide (Table 3) = 2.33/3 = 0.78. Millimoles of choline degraded to yield 25% of the dimethylamine excretion of 0.68 methyl meq (Table 3) = (0.68 x 0.25)/2 = 0.09. Total degradation of choline = 0.78 + 0.09 = 0.87 = 0.9 mmoles. tT. Sargent, personal communication to the authors (1974).
LABILE METHYL
729
BALANCES
Reactions Fig. 3. possibly involved in methyl-group oxidations. The lefthand cycle is that proposed by Mackenzie and Frisell.” The particcompounds ipating in the portion of this cycle between serine and choline may be the phosphatidyl derivatives. The righthand cycle is that proposed by glumenstein and Williams?Q
ETHANOLAMINE
METHYL.
0
bodily CO* pools. The extent of oxidation of choline methyl will also be underestimated by measurement of respiratory 14C02 production for much the same reasons, since the one-carbon fragments formed by oxidation of this compound enter the same metabolic pool as those produced by oxidation of methionine methyls (Fig. 3). An alternative index of the rate of methyl-group oxidation is provided by measurements of sarcosine excretion in hypersarcosinemic patients. Such patients are presumed to lack activity of sarcosine dehydrogenase,53 a presumption which has been confirmed by hepatic enzyme assay in one case.54 Both the enzyme data54 and the fact that for this patient, in contrast to the normal,s3 60”/0-80% of sarcosine loads were recovered in the urine in the first 24 hr* indicate that the metabolic block in this patient was complete enough to ensure that she metabolized little sarcosine. Since there is little or no sarcosine in the plants5 or animals6 materials found in normal diets, and since the normal human excretes little, if any, sarcosine (O-0.02 mmole/24 hr),53 the sarcosine excreted by this sarcosine dehydrogenase-deficient patient provides a measure of the amount of sarcosine endogenously formed and subsequently metabolized by sarcosine dehydrogenase in the normal human. At age 6, under basal conditions, the girl in question excreted 5.3-7.9 mmole sarcosine in her daily urine.53 At age 12, a single daily urine contained 2.9 mmole sarcosine.1 The few additional published values for sarcosine excretion in hypersarcosinemic patients are all derived from boys 2 yr old or younger. These values range from 0.9 mmoles/24 hr (age 3 mo) to 5.6 mmoles/24 hr (age 2 yr).37,57,58Although clearly of interest for our present purposes, no data on the effects of variations in methionine, choline, cystine, or glycine intakes on sarcosine excretion have been reported for hypersarcosinemic patients. Thus, the rather fragmentary data presently available suggest that the normal human will probably metabolize in the range of 5 + 3 mmoles of methyl groups daily by this pathway. *T.
Gerritsen,
personal
communication
to the authors
(1974).
730
MUDD
AND POOLE
These oxidized methyl groups may derive from either the labile methyl pool (methionine and one of the choline-betaine methyls) or from the biologically nonlabile methyls of choline-betaine which become the methyl of sarcosine. On the equilibration diets, our male subjects were absorbing approximately 3.6 mmoles of choline. The fate of this compound is not known with certainty, but complete conversion to sarcosine would account for the major portion of the 5 + 3 mmoles of this compound which are expected to be formed and metabolized in the normal human each day. If so, under these dietary conditions, relatively little of the oxidized methyls would have derived from labile methyls, a conclusion which agrees with the results of the measurements of respiratory 14COz which indicate that somewhat more than 0.6 mmoles of methionine methyl were oxidized each day. On the experimental diets, on the other hand, the subjects received many fewer nonlabile dietary methyls (Table 2), so that, if methyl-group oxidation had continued unabated, a large portion would have derived from labile methyls. However, the dietary conditions of decreased methionine and lack of choline are those which, in the rat, decreased the rate of 14C02 production from “CH,-methionine. Thus, it seems probable that total methyl-group oxidation may have been severely curtailed during the experimental diets and that, again, only small amounts of labile methyls would have been accounted for by oxidation.
Pol_vamine Biosynthesis Additional methionine will be utilized for spermine and spermidine biosynthesis. The three-carbon moieties of these polyamines are formed from decarboxylated S-adenosylmethionine. 59 The methyl-containing product of these reactions if 5’-methylthioadenosine, which may subsequently be subjected to phosphorolysis and hydrolysis to yield methylthioribose@’ (Fig. 1). The metabolic fate of this compound is unknown, but its methyl group may be presumed to be the labile methyl pool. In view of these uncertainties as to the methylated excretory product(s) formed as a result of polyamine synthesis, the demand on the methionine pool for polyamine synthesis was estimated from the turnover rate: The total spermine in the adult human body (calculated by use of the concentrations reported and using normal organ weights6*) is somewhat less than by Tabor and Tabor, 4 mmoles. We are not aware of comparable data for spermidine. However, the comparative spermidine and spermine contents of a variety of mammalian tissue@ indicate that the total body spermidine may be of the same order of magnitude as that of spermine. Spermidine of rat liver and brain has been estimated to turn over with a half-life of about 4 days63 or longer.@ If total human body spermidine turns over at a similar rate, approximately 0.5 mmole of methionine would be utilized daily in the course of the biosynthesis of this polyamine. This may be a considerable overestimate, since the human turnover rate is likely to be lower, and since dietary intake of spermidine has not been taken into account. Since the turnover of spermine is markedly slower than that of spermidine,63 the diversion of methionine for spermine biosynthesis is likely to be so low as to be negligible for our present purposes. Thus, the total daily
LABILE
METHYL
731
BALANCES
polyamine synthesis in the adult human may be taken as 0.5 mmole/day or less. There will be a corresponding demand for methionine. In distinction to utilization of methionine for methylation, or for methyl-group oxidation, polyamine synthesis also involves a net loss of the homocysteinyl moiety of methionine. Methionine following this pathway will contribute neither to transmethylation nor to the sulfur conservation cycle. Balances and Cycles
Sufficient data are now available to permit tentative calculations of labile methyl-group balances during the present experiments. The pertinent values are summarized in Table 4. Apparently, for normal humans, the demand for labile methyl groups is determined to a major extent by the requirement for creatine-creatinine formation. Creatinine is formed nonenzymically in proportion to the amount of phosphocreatine and creatine stored in muscle.65 The result is to place upon the normal adult human a relatively large and inflexible demand for labile methyl groups. In males, with larger muscle masses, this demand by itself may outweigh the labile methyls absorbed from an ordinary diet. For females, creatinine formation is less and may possibly be balanced by the Table 4.
Tentative
labile Methyl Ralances on Several Dietary Regimens M&r
Equilibration
Females
Experimental
A
Experimental
Diet
(meq
Equilibr.tion
B lobile
methyl/24
Experimental
A
Experimental
B
hr)
Inputs Methionlne’
9.9
4.7
7.8
9.2
45
Cholinet
3.6
o-o.4
o-o.5
25
o-o.4
Total
13.5
7.8-8.3
4.7-5.1
11 7
a2
I
o-o.
4 5-4.9
8.2-8
3
0”tnowr Creatin8net Other
Polyomine
15.8
15.4
10.3
10.6
1.9
1.8
2.0
2.6
2.1
16
0.5
05 ?
05
0.3
05
>0.6
0.5 ?
>0.6
?
?
18.6
18.1
17.9
14.0
13.2
120
5.1
13.0
9.6
23
8.3
37
1.9
3.9
2.3
I5
3.0
14
ryntherir
Oxidationq Total
15.6
methylations$
occwnted
Mi”irn”rn
for
ertimoter
of average
Methylneagenerirll Cyclea
99
per homocysteinyl
moiety** Per cent homocyr+eine remsthyhtedt
*From tVolues
57
74
33
67
29
2.
from
certainty these
47
Table
Table
2, excluding
in the values
during
the
portion
the experimental
of choline diets
degraded
reflects
by intestinal
uncertainties
bacteria
as to bacterial
(see text).
The
degradation
un-
under
conditions.
$Values present
during
during
perimental
diets
jjlncludes choline,
creatine
(Table
discussion
**Total
2) and
all
or from
Table
for
2. In order
equilibration
to exclude
diets
are
dietory
means
sources
of all
values
of
creatinine
during
the
ex-
methyloted oxide,
compounds
and
the
listed
methyls
of
in
Table
dimethylomine
3,
except arising
for from
methionine, bacterial
in text.
methyl
output
minus
tronsmethylations
-
from
listed
glycine.
+
maximum time-averaged
planation. t t 100
taken
values
trimethylamine
of choline
(IMinimum
diets
diets,
(see text).
trimethylomine,
degrodotion qSee
experimental
equilibration
(1 OO/averoge
No.
cycles).
labile
methyl
intake.
homocysteinyl
moiety
available.
See
text
for
further
ex-
732
MUDD
AND
POOLE
normal labile methyl intake. In view of the quantitative importance of creatine formation as a fate for labile methyl groups, it would be of interest to reinvestigate factors affecting the activity of guanidinoacetate methyltransferase (E.C. 2.1.1.2). The catalytic properties of this enzyme have received little experimental attention since the pioneering studies of Cantoni and his collaborators@ 20 yr ago. A second conclusion which emerges is that when the labile methyls required for methylations in addition to that of guanidinoacetate are taken into account, endogenous formation of methylated compounds must normally exceed the dietary intake of labile methyl groups. Methyl group neogenesis must then normally be required. Conversely, the homocysteine conservation cycle must normally be operative. For example (Table 4), for the male subjects receiving the equilibration diets, 9.9 mmoles of homocysteine moieties were made available daily, of which up to 0.5 mmoles were removed during the course of each 24 hr for polyamine synthesis, Neglecting minor alternative pathways for degradation of methionine and homocysteine, and averaging over time, this left available approximately 9.7 mmoles of homocysteine which cycled sufficiently, so that at least 18.0 mmoles of methylation occurred from S-adenosylmethionine. More transmethylation may have occurred to the extent that oxidation of methionine methyl by way of sarcosine has been underestimated, and that endogenously formed methylated compounds lost in urine or stools were not taken into account. Each homocysteine moiety must then have cycled on the average at least 1.9 times. (Since each of the uncertainties specified here would lead to underestimation of the extent of cycling, the value of 1.9 is a minimal one.) Expressed in another way, under these conditions at any moment the chances were that at least 47% of the available homocysteine was being reconverted to methionine, while the remainder was being diverted to cystathionine.* As a third conclusion, it is clear that when methionine and choline intakes are curtailed, methyl neogenesis becomes the dominant source of labile methyls. Under the particular conditions of experimental diet A, the average homocysteinyl moiety passed through the sulfur conservation cycle at least 3.9 times in the male subjects, and 3 times in the females (Table 4). The present data are compatible with, although they do not prove, the possibility that methionine intake controls methyl neogenesis so that a reduction in methionine ingestion leads to an enhancement of de novo methyl-group formation. Possible mechanisms to mediate such an effect are suggested by the observations that in rat liver S-adenosylmethionine may inhibit the activity of methylenetetrahydrofolate reductase,68 and that dietary methionine may decrease the activity of ‘Nmethyltetrahydrofolate-homocysteine methyltransferase69 and prevent the accumulation of 5N-methyltetrahydrofolate.70 The values summarized in Table 4 are merely first approximations. Improvements could be made by more complete measurements of methylated excretory products and especially by determination of the extent of oxidation of labile
*This value is within the range of 40”-60% of homocysteine converted each cycle, reported as a preliminary estimate for normal rat liver.67
to cystathionine
during
LABILE
METHYL
733
BALANCES
and nonlabile methyls under a variety of conditions. The specific results obtained apply only to the dietary conditions employed and may have been influenced to an unknown extent, for example, by the very low cystine content of the experimental diets. Nevertheless, the present calculations appear to be sufficient to outline the main avenues of labile methyl-group metabolism in the adult human and to provide a point of departure for assessment of the effects of genetic, nutritional, pharmacologic, or hormonal factors which alter the quantitative relationships between these avenues. ACKNOWLEDGMENT The authors wish to thank Dr. G. L. Cantoni for his interest in this work, MS B. Conerly for performing amino acid analyses, MS E. Bou for her help in planning and analyzing the diets. and MS K. Pettigrew
for analyzing
the data for statistical
outliers.
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LABILE
METHYL
735
BALANCES
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